Cysteine Profiling Service using LC-MS/MS

Introduction: Why Cysteine PTMs Matter

Cysteine stands out among proteinogenic amino acids because its thiol (-SH) side chain is exquisitely redox-sensitive, acting as both a catalytic nucleophile and a molecular switch that responds to oxidative cues. A single cysteine post-translational modification (PTM)—be it reversible S-nitrosylation or the formation of an intramolecular disulfide—can realign a protein's conformation, alter its interaction landscape, and ultimately reprogram entire signaling cascades. In drug-discovery settings, these redox-driven switches modulate target engagement, off-target liabilities, and even compound stability, making cysteine PTMs a powerful—but often under-measured—layer of functional regulation.

Despite their importance, cysteine modifications are notoriously transient and present at low stoichiometry, meaning traditional proteomics workflows miss a substantial fraction of the actionable data. Purpose-built cysteine post-translational modification profiling therefore provides a sharper lens for researchers investigating stress responses, kinase regulation, or covalent inhibitor mechanisms. By capturing the dynamic redox landscape instead of static protein abundance alone, scientists gain a more predictive understanding of cellular behavior—insights that are increasingly pivotal for academic discovery and pre-clinical R&D alike.

Common Cysteine Modifications at a Glance

Cysteine's thiol is chemically versatile, giving rise to a spectrum of post-translational modifications (PTMs) that act as rapid "switches" in redox and signaling networks. Below are five of the most biologically prominent—each detectable through our cysteine profiling workflow.

Why it matters for your project: Each PTM leaves a unique chemical "fingerprint" that our selective enrichment chemistries and high-resolution LC-MS/MS can capture. Whether you need S-nitrosylation analysis for oxidative stress models or palmitoylation mapping

Research & Drug-Discovery Challenges

High-quality cysteine PTM data are difficult to obtain because the chemistry we're trying to capture is both fleeting and sparse. In whole-cell lysates,<5 % of any given cysteine population may carry a specific modification at steady state; sample handling further skews this low stoichiometry by introducing thiol oxidation artifacts or causing labile PTMs (e.g., S-nitrosylation) to collapse before analysis. Even when the modification survives, a single reactive probe rarely labels every accessible cysteine: head-to-head comparisons show pronounced probe- and warhead-specific biases that leave swaths of the "cysteinome" invisible to any one workflow.

Key Pain Points for Researchers

Low stoichiometry & dynamic range – Modified cysteines are often present at sub-percent levels, demanding highly efficient enrichment and ultra-sensitive LC-MS/MS.

Probe coverage & selectivity – Comparative datasets (e.g., CysDB) reveal that different iodoacetamide- or dimedone-based chemistries capture overlapping but non-identical site sets, complicating global profiling studies.

Sample-induced artifacts – Air exposure, residual metal ions, or non-ideal buffers can oxidize free thiols, masking true in-vivo PTM states if not rigorously controlled.

MS/MS fragmentation pitfalls – Click-based capture tags can fragment unpredictably, reducing peptide ID rates unless acquisition methods and search algorithms are optimized (Yan et al., 2022). We minimise this issue by adopting short-linker biotin-DBCO tags and optimised library-search parameters.

Bioinformatic localization – Distinguishing closely spaced cysteines or multiple oxidative states on the same residue requires specialized scoring models and manual validation checkpoints.

Because of these hurdles, many labs see high false-negative rates or irreproducible hit lists when attempting in-house assays. A dedicated Cysteine Profiling Service—equipped with modular probe panels, inert-atmosphere sample prep, and purpose-built informatics—solves these challenges and delivers reliable, quantifiable insights that directly inform covalent inhibitor design, stress-response studies, and redox signaling projects.

Service Overview — Creative Proteomics Cysteine Profiling Platform

Creative Proteomics has built a Cysteine Profiling Service specifically for redox-sensitive residues, combining chemistry-matched enrichment probes with state-of-the-art LC-MS/MS cysteine analysis and bespoke bioinformatics. The platform is modular, letting clients interrogate a single PTM (e.g., S-nitrosylation) or run a broad "cysteinome" screen in one streamlined project.

When your project requires multi-layered PTM insight, the cysteine platform plugs directly into our broader [Protein Post-Translational Modification Analysis Service] for phospho-, acetyl-, or ubiquitin-level context—no extra sample prep needed.

Technology & Workflow — Step-by-Step

Below is the standardized cysteine enrichment workflow we apply to every project. Each stage is performed under rigorously controlled, oxygen-limited conditions to preserve labile thiol chemistry and maximize PTM site mapping depth.

By integrating stringent thiol preservation, chemistry-selective capture, and sub-ppm Orbitrap MS acquisition, our pipeline consistently identifies >25 k unique cysteine sites with high localization confidence—transforming the traditionally elusive cysteine redox landscape into actionable, quantitative datasets.

Advantages of Partnering with Creative Proteomics

1. Unrivalled Cysteine-Modification Coverage

Profile seven major reversible chemistries in one project: S-palmitoylation and other thio-acylations, S-nitrosylation, S-glutathionylation, S-sulfenylation (-SOH), S-sulfination (-SO₂H), persulfidation (-SSH), global reversible oxidation, plus free-thiol mapping.

2. Dual Labelling Strategies for Depth and Flexibility

Biotin-exchange workflow (streptavidin enrichment) — no cap on sample number, very low reagent cost, and stronger affinity than antibody-based pull-downs, giving the deepest site recovery for discovery screens.

TMT multiplexing — up to 16 conditions in one LC-MS injection for accurate relative quantification when sample numbers are defined.

Our team selects—or combines—these chemistries to match study size, quantitative needs and budget.

3. Ultra-High Sensitivity, Validated by Site-Level FDR

Orbitrap Exploris 480 and timsTOF HT routinely quantify > 25 000 unique cysteine sites at ≤ 1 % site-level FDR, capturing low-stoichiometry events such as covalent-drug engagement or stress-induced redox switches.

4. Control-Driven Experimental Design

Each biological sample is paired with an internal control to eliminate system bias. Strict localisation scoring and a global 1 % false-discovery filter ensure every reported site is robust enough for publication review.

5. Rapid Turnaround with Expert Guidance

EvoSep high-throughput LC, automated data pipelines and a dedicated Ph.D. project manager keep timelines short and communication seamless—from probe choice to figure-ready deliverables.

6. One-Stop PTM Expansion and Scalable Pricing

The cysteine workflow dovetails directly with our phospho-, acetyl- and ubiquitin-omics services, enabling facile systems-biology add-ons. Pricing tiers cover single samples, TMT multiplexes or 96-well plate screens, so you can start lean and scale fast.

Sample Requirements & Submission Guidelines

Buffer compatibility

Avoid glycerol > 5 %, strong detergents (e.g., SDS > 0.1 %), and high salt > 250 mM.

Do not add reducing agents (DTT, TCEP) or alkylators (IAA, NEM); our workflow introduces controlled labelling.

Packaging & Shipping

• Aliquot samples to minimise freeze-thaw cycles (≤ 500 µL or ≤ 500 mg per tube).
• Label tube tops and sides with sample ID, matrix, and weight/volume.
• Freeze at −80 °C and ship overnight on dry ice (≥ 5 kg for international parcels).
• Include the completed Cysteine-PTM Sample Submission Form inside the package.

Turnaround Options remain unchanged (Standard / Priority / High-throughput); the project clock starts once samples pass QC for weight, protein concentration and buffer compatibility.

Deliverables & Data Interpretation Support

Site-level validation advice (e.g., targeted PRM assays, mutagenesis controls).

Integration support with our global PTM or whole-proteome datasets when broader context is required.

DEMO Results Snapshot

Frequently Asked Questions (FAQs)

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Case Study Spotlight — From Cysteine Oxidation Maps to Drug-Target Insight

• Overview
• Results
• Conclusion
• Why it matters

A recent investigation in Journal of Translational Medicine used high-coverage cysteine redox proteomics to interrogate pancreatic-cancer biology (Tan et al., 2024. DOI: https://doi.org/10.1186/s12967-024-05068-z). Researchers compared the PDAC cell line PANC-1 with non-tumorigenic HPDE6c7 cells using an iodoTMT-based enrichment strategy followed by Orbitrap LC-MS/MS—an approach that mirrors our own Cysteine Profiling Service workflow.

Project Snapshot	Outcome
Scope	Triplicate, label-based quantitation of reversible cysteine oxidation across two cell states
Depth	> 4,300 oxidized sites on > 2,100 proteins; 1,715 sites showed significant differential oxidation
Key Finding	Selective oxidation of HIF-1 pathway regulators—notably prolyl hydroxylase PHD2—suggested that redox control, rather than expression level, drives hypoxia signaling in PDAC
Drug-Discovery Relevance	Oxidation-sensitive cysteines clustered around HIF-1 regulatory nodes, flagging potential covalent-allosteric sites for small-molecule intervention and providing a mechanistic read-out for evaluating redox-modulating chemotypes

Target Validation: Oxidation hotspots on PHD2 and VHL adaptor proteins pointed to redox gating of HIF-1α degradation—an actionable vulnerability for hypoxia-targeted therapies.

Biomarker Development: Cysteine-specific oxidation ratios served as pharmacodynamic markers to track the efficacy of emerging HIF-1 inhibitors in follow-up compound screens.

Combination Concepts: Overlaying cysteine oxidation changes with phosphoproteomic data suggested synergistic inhibition of both redox buffering and kinase-signaling arms, guiding rational drug-combination design.

This study illustrates how deep-scale cysteine PTM profiling can surface functional drug-response markers that would remain invisible to transcriptomics or total-proteome surveys alone.

Publication

Here are some publications in PTMs Proteomics research from our clients:

References

1. Yanzheng Meng, Lin Zhang, Laizhi Zhang, Ziyu Wang, Xuanwen Wang, Chan Li, Yu Chen, Shipeng Shang, Lei Li, CysModDB: a comprehensive platform with the integration of manually curated resources and analysis tools for cysteine posttranslational modifications, Briefings in Bioinformatics, 2022;, bbac460, https://doi.org/10.1093/bib/bbac460
2. White MEH, Gil J, Tate EW. Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics. Cell Chem Biol. 2023 Jul 20;30(7):828-838.e4. DOI: 10.1016/j.chembiol.2023.06.021
3. Biggs, G.S., Cawood, E.E., Vuorinen, A. et al. Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics. Nat Commun 16, 73 (2025). https://doi.org/10.1038/s41467-024-55057-5
4. Boatner LM, Palafox MF, Schweppe DK, Backus KM. CysDB: a human cysteine database based on experimental quantitative chemoproteomics. Cell Chem Biol. 2023 Jun 15;30(6):683-698.e3. https://pubmed.ncbi.nlm.nih.gov/37119813/
5. Yan T, Palmer AB, Geiszler DJ, Polasky DA, Boatner LM, Burton NR, Armenta E, Nesvizhskii AI, Backus KM. Enhancing Cysteine Chemoproteomic Coverage through Systematic Assessment of Click Chemistry Product Fragmentation. Anal Chem. 2022 Mar 8;94(9):3800-3810. DOI: 10.1021/acs.analchem.1c04402